The advancement in biological cellular process technologies allows the reprogramming of matured cells such as somatic cells to an embryonic-like state. These “induced pluripotent stem cells” (iPSCs) enable a new patient-specific or “personalized” medicine paradigm in 1) autologous regenerative medicine, 2) disease cell lines from patient cells for more predictive assays in drug discovery and basic research, and 3) patient-specific cell generation for personalized diagnostic and drug efficacy or adverse effects testing. To achieve the promise of iPSC technology requires the controlled differentiation of iPSCs to specific lineages.
However, the yields of reprogramming and differentiation are both very low. For any given matured cells under reprogramming, it is not at all certain that any significant number of iPSCs will be produced. Similarly, for any given iPSC colony under differentiation, it is not at all certain that any significant number of target cells will be produced.
To mitigate the low yield and the lack of certainty, state-of-art protocols require a large number of samples to carry out the full reprogramming process, and selection of more iPSC colonies for expansion and characterization than is desirable. For the differentiation process, iPSC lines are selected in an ad-hoc fashion and a large number of stem cells are used for differentiation in the hope of generating a sufficient quantity of target cells. This approach is time consuming and expensive as reflected by the prices of iPSC derived cells costing more than 10× the price of adult human primary fibroblasts.
For the reprogramming process, iPSC colony selection is largely done manually with additional staining such as TRA-1-60 surface marker or using viral-GFP silencing when available. However, additional staining is not desirable in practical clinical laboratory settings. Therefore, it is desirable to make the selection using non-invasive imaging modalities such as phase contrast images. There are computer image recognition methods that are developed to determine between iPSCs and Non-iPSCs. The prior art methods are based on fixed images acquired near the end of reprogramming. Therefore, many colonies have to go through the full course of reprogramming which is costly and inefficient. To date, there are no effective image recognition methods that can predict the outcomes of the differentiation process.
Reprogramming for induced pluripotent stem cells (iPSCs) is a slow process. It takes around 4 weeks for reprogrammed cells to reach a pluripotent state. In the process many colonies are formed, but only a small number of them are true iPSC colonies. The current practice selects true iPSC colonies near the end of the reprogramming process (harvesting time). The selection could be done using morphological criteria from the microscopy images of the colonies either by human eye or by computer image analysis. Another selection method uses the images of additional staining such as TRA-1-60 surface marker or viral GFP-silencing to assist the determination of iPSC colonies from non-iPSC ones. In addition, the iPSC PCR Array is used to analyze multiple biologically validated pluripotency biomarkers to distinguish fully reprogrammed iPSC colonies from partially reprogrammed ones. There are many issues with the current practices as listed below:    1. Morphological criteria are non-specific at harvesting time. There are many non iPSC colonies with similar appearance to iPSC colonies that could confuse the selection;    2. At harvesting time, the grown colonies could merge or interfere with each other. Therefore, true iPSC colonies could be mixed with or contaminated by non-iPSC colonies;    3. Colonies are not necessarily synchronized in the reprogramming process. At a fixed harvesting time, some colonies may not have fully reprogrammed and some may have passed the iPSC state and have spontaneously differentiated;    4. It is unnecessarily costly to select multiple iPSC colonies. More accurate, specific selection could reduce the number of colonies required;    5. It is wasteful to process the large number of non-productive colonies through the whole reprogramming process.
We discovered an early kinetic pattern of colony formation for cells undergoing reprogramming, within the first 72 hours after a colony is first discernible. The kinetic pattern can reliably predict which cells will most likely progress to become induced pluripotent stem (iPS) cell colonies (See Hendrik et al “Assessment of an imaging protocol for real-time selection of human iPSC colonies using live cell microscopy and image recognition software”, poster presentation at the ISSCR 10th Annual Meeting, June 2012). Similar early kinetic patterns can also accurately predict which colonies will most likely differentiate with good cardiomyocyte yield (Alworth et al., “Real-time scoring of human iPSC differentiation potential using live-cell microscopy and image recognition software”, poster presentation at the ISSCR 10th Annual Meeting, June 2012). Studies in the field have focused on late colony morphology at around three weeks, or the use of surface markers for iPS selection. The quantification of colony formation dynamics could generate powerful kinetic image features that can be used reliably for real-time colony outcome prediction in a much earlier stage of reprogramming. The outcomes include not only iPSCs formation but also prediction of differentiation yield.